The proposal focuses on G protein-coupled receptor (GPCR) signaling in the fungal pathogen Cryptococcus neoformans, which infects the lung and often disseminates to the central nervous system to cause life- threatening meningitis. There is an urgent unmet medical need for developing new drug targets for better treatment of cryptococcal infection. To meet this need, it is critical to better understand the disease mechanisms by which the fungus senses host signals and adapts to the hostile host environment. One such adaptation strategy during infection is to dramatically enlarge yeast cell size, a novel cell morphogenesis called cell gigantism or titan cell formation. Fungal cell gigantism during lung infection has been recently recognized as a new virulence factor that is required for disease initiation and dissemination. Despite its importance in fungal pathogenesis, the host signals and fungal receptors for titan cell induction remain unknown. Studies from the applicant demonstrated that the Gpa1 (G protein subunit) G protein signal transduction pathway is involved in the production of titan cells. A novel G protein-coupled receptor (GPCR), Gpr5, has been identified as being essential for promoting titan cell formation. These new discoveries led to the central hypothesis that fungal cells sense host specific signals through the Gpr5 orphan receptor, which then activates Gpa1-mediated signaling to regulate fungal cell morphogenesis. The long-term goal is to understand the molecular basis of cryptococcal cell gigantism in order to provide the mechanistic details needed to develop new antifungal agents that interfere with GPCR function and inhibit titan cell formation during Cryptococcus infection. Guided by strong preliminary data, the hypothesis will be tested by two specific aims: 1) Define the function of Gpr5 in G-protein signaling activation and fungal titan cell regulation, and 2) Identify and characterize host signals required for Gpr5-mediated fungal cell gigantism. Under the first aim, strains having various levels of Gpr5 activity will be used to determine the role of Gpr5 in fungal titan cell production and pathogenesis in murine models, and to characterize how Gpr5 regulates the activation of Gpa1 as measured by cellular cAMP production. Under the second aim, a spent medium that can stimulate titan cell formation in vitro will be used to identify active compounds that are responsible for Gpr5-dependent cell gigantism. A Saccharomyces-based heterologous expression system will be used to analyze potential ligands for Gpr5 activation. The approach is innovative, because we have developed an in vitro system for titan cell formation that allows us to identify host factors responsible for cell gigantism. The proposed research is significant, because it aims to understand the molecular basis of the host-pathogen interaction during the development of virulence factors, which is critical for fungal pathogenesis. Ultimately, such knowledge will facilitate efforts to design new anti-fungal agents to perturb GPCR signaling in a therapeutically beneficial manner.